DNA cloning

DNA cloning - Strategies

1. DNA Cloning methods Presented By, S.Divya , 2017600805 TAMIL NADU AGRICULTURAL UNIVERSITY , COIMBATORE- 641003

2. DNA CLONING DNA cloning is a technique for reproducing DNA fragments.  It can be achieved by two different approaches:  ▪ cell based (in vivo) ▪ using polymerase chain reaction (PCR) (in vitro).    

3. CLONING PROCESS Gene of interest is cut out with RE Host plasmid is cut with same RE Gene is inserted into plasmid and ligated with ligase New plasmid inserted into bacterium (transform)

4. PLASMID CLONING STRATEGY Involves five steps : Enzyme restriction digest of DNA sample. Enzyme restriction digest of DNA plasmid vector. Ligation of DNA sample products and plasmid vector. Transformation with the ligation products. Growth on agar plates with selection for antibiotic resistance .

5. STEP 1- RE DIGESTION OF DNA SAMPLE Pst 1 Pst 1 Pst 1 Pst 1 Pst 1

6. STEP 2- RE DIGESTION OF PLASMID DNA

7. STEP 3- LIGATION OF DNA SAMPLE AND PLASMID DNA

8. Clone a foreign DNA into the Pst I site of pBR322 Cut the vector to generate the sticky ends Cut foreign DNA with Pst I also – compatible ends Combine vector and foreign DNA with DNA ligase to seal sticky ends Now transform the plasmid into E. coli 4- 8 Cloning using pBR322

9. STEP 4- TRANSFORMATION OF LIGATION PRODUCTS The process of transferring exogenous DNA into cells is call “transformation ” There are basically two general methods for transforming bacteria. The first is a chemical method utilizing CaCl2 and heat shock to promote DNA entry into cells. A second method is called electroporation based on a short pulse of electric charge to facilitate DNA uptake.

10. CHEMICAL TRANSFORMATION WITH CALCIUM CHLORIDE

11. TRANSFORMATION BY ELECTROPORATION

12. STEP 5 . GROWTH ON AGAR PLATES

13. If new DNA is inserted at that Pst I restriction site, it inactivates the gene for ampicillin resistance. Plasmid then has gene for tetracyclin resistance, but not for ampicillin. This can be used to select for host cells with new DNA. STEP 5

14. TYPES OF CLONING VECTORS

15. PLASMID VECTORS Plasmid vectors are used to clone DNA ranging in size from several base pairs to several thousands of base pairs (100bp -10kb). ColE1 based, pUC vehicles commercially available ones, eg pBR322, pBlueScript

16. Disadvantages using plasmids Cannot accept large fragments Sizes range from 0- 10 kb Standard methods of transformation are inefficient

17. BACTERIOPHAGE LAMBDA Phage lambda is a bacteriophage or phage , i.e. bacterial virus, that uses E. coli as host. Its structure is that of a typical phage: head, tail, tail fibres. Infection: lambda tail fibres adsorb to a cell surface receptor, the tail contracts, and the DNA is injected. The DNA circularizes at the cos site, and lambda begins its life cycle in the E. coli host.

18. BACTERIOPHAGE LAMBDA

19. COSMID VECTOR Purpose: 1. Clone large inserts of DNA: size ~ 45 kb Features: Cosmids are Plasmids with one or two Lambda Cos sites. Presence of the Cos site permits in vitro packaging of cosmid DNA into Lambda particles

20. Yeast Artificial Chromosomes

21. Yeast Artificial Chromosomes Purpose: Cloning vehicles that propagate in eukaryotic cell hosts as eukaryotic Chromosomes Clone very large inserts of DNA: 100 kb - 10 Mb Features: YAC cloning vehicles are plasmids Final chimeric DNA is a linear DNA molecule with telomeric ends: Artificial Chromosome

22. RETROVIRAL VECTORS Retroviral vectors are used to introduce new or altered genes into the genomes of human and animal cells. Retroviruses are RNA viruses. The viral RNA is converted into DNA by the viral reverse transcriptase and then is efficiently integrated into the host genome Any foreign or mutated host gene introduced into the retroviral genome will be integrated into the host chromosome and can reside there practically indefinitely. Retroviral vectors are widely used to study oncogenes and other human genes.

23. SHUTTLE VECTORS Shuttle vectors can replicate in two different organisms, e.g. bacteria and yeast, or mammalian cells and bacteria. They have the appropriate origins of replication. Hence one can clone a gene in bacteria, maybe modify it or mutate it in bacteria, and test its function by introducing it into yeast or animal cells.  

24. PCR cloning strategies Cloning methods for PCR products are divided into three types: ( i ) blunt-end cloning (ii) sticky-end cloning (iii) T-A cloning

25. T-A Cloning When DNA fragments are generated Taq polymerase adds 1 or 2 extra adenines onto the end of 3’ end of blunt ds DNA Several commercially available kits take advantage of this ability Use a plasmid vector with thymidine residues linked onto the 3’ ends of linearised plasmid DNA

26. The Topo TA Cloning Process

27. Why Are We Topo Cloning? PCR generates the exact gene fragment we want to clone. PCR products and no other DNA are ligated into the Topo vector by the topoisomerase. Ligation is highly efficient (as high as 90%). Selection of transformants is highly effiecient A very large number of white colonies is generated

28. Reference S.S.Purohit ,Biotechnology Fundamentals and Applications,Fourth Edition AGROBIOS Publishers